COMPOSITIONS FOR USE IN THE TREATMENT AND PREVENTION OF ULCERATIVE COLLITIS

FIELD OF THE INVENTION

The invention provides compositions and methods for treating and preventing ulcerative colitis (or colitis ulcerosa). The compositions and methods of the invention are based on a novel use of thiocyanate or isothiocya- nate comprising compounds and other compounds useful for enhancing thiocyanate or isothiocyanate levels in the serum and/or in the colon.

BACKGROUND OF THE INVENTION Ulcerative colitis, which is one of the two major versions of the so called "inflammatory bowel disease"

(IBD) is a chronic, recurrent disease characterized by inflammation involving the colon. Crohn's disease (the other major version of IBD) is a chronic, recurrent disease where inflammation can occur in any part of the gastrointestinal tract. Despite many years of intense research, the pathogenesis of both major versions of IBD is still poorly understood. Understanding the molecular events, which lead to the pathogenesis of IBD would be essen- tial for the development of more effective, new therapeutic strategies. There are many observations suggesting that increased production of reactive oxygen species (ROS) in the intestinal mucosa has an important role in the development of IBD ( ^1>2 ). The production of a high amount of ROS was observed in clinical biopsy samples of patients with IBD ( ³ ' ⁴ ). ROS, specifically superoxide (O ₂ ^" ) can also react with nitric oxide (NO ) and form the highly reactive metabolite, peroxynitrite (OONO ^~ ). Peroxynitrite then induces cytotoxic processes, partially through the nitration of tyrosine residues of proteins. The formation of nitro tyro sine residues was demonstrated in the biopsy samples of IBD patients ( ⁵ ).

Despite intensive research effort there are still no effective therapies for these diseases. Present therapy includes treatment with corticosteroids and 5-aminosalicylic acid derivatives. Ulcerative colitis is characteristically a disease of non-smokers and the disease frequently develops in patients after they quit smoking ( ⁶ ). No similar observation exists in Crohn's disease. The explanation for the "beneficial" effect of smoking in ulcerative colitis has so far been unknown. Nicotine was suggested to be responsible for this effect, but recent studies did not prove its efficiency in patients with ulcerative colitis ( ⁷ )

The alteration of the functioning of the peroxidase - halide - hydrogen peroxide host defense system has recently been proposed to be applied in the treatment of a number of inflammatory disorders where the presence of some peroxidase activity has been demonstrated at the site of inflammation, either by tissue specific expression (i.e. in the case of cystic fibrosis: US 6,702,998) or by specific expression by eosinophil granulocytes migrating to the inflammation site (WO0241885). The said host defense system, when functioning properly, is a potent mechanism participating in the antimicrobial protection of higher organisms, nevertheless, the potentially self-toxic reactive compounds produced by the system may also be involved in the development of undesired in- flammatory disorders.

In the human body, the peroxidase - halide/pseudohalide - hydrogen peroxide host defense systems synthesize potent antimicrobial compounds through the oxidation of halide/pseudohalide ions into reactive species. The major components of the human peroxidase - halide/pseudohalide - hydrogen peroxide host defense systems are as follows.

Peroxidases

Lactoperoxidase (LPO) is a member of the family of mammalian peroxidases. Peroxidases are heme- containing enzymes, which use hydrogen peroxide H ₂ O ₂ to oxidize different substrates ( ⁸ ). The structure of peroxidases is highly conserved across the plant and animal kingdom. They all contain specific amino acids in highly conserved locations and these amino acids are responsible for the binding of heme ( ⁸ ). Mammalian peroxidases include myeloperoxidase (MPO), eosinophil peroxidase (EPO), thyroid peroxidase (TPO), lactoperoxidase (LPO) and peroxidasin. LPO was first described in milk where it is present in high concentration ( ⁹ ). LPO is also a component in other exocrine secretions including the saliva and bronchial secretion ( ¹⁰ ). The presence of LPO in tear and cervical fluid was also suggested, although genetic or immunological evidence were not pro- vided. LPO is produced by exocrine glands, which include salivary glands, mammary glands and submucosal glands of the trachea and bronchi. Under physiological conditions LPO is the dominant peroxidase in the airways. However, when inflammation occurs neutrophil or eosinophil granulocytes migrate to these tissues and these cells contain high amount of MPO or EPO.

Hydrogen peroxide (H ₂ O ₂ ) For the EPO- and MPO-catalyzed reactions, the phagocytic NADPH oxidase provides hydrogen peroxide.

This H ₂ O ₂ -producing system is effectively activated by different inflammatory cytokines and bacterial products ( ^π ). The H ₂ O ₂ -SOUTCe of the LPO system is less clear although recent data suggest that Dual oxidases, which are present on mucosal surfaces, provide H ₂ O ₂ for the LPO-catalyzed reactions ( ¹² ' ¹³ ). It was recently described the Dual oxidases (Duox enzymes) are also effectively upregulated by inflammatory cytokines including interferon- gamma, suggesting that during airway inflammation Duox-based H ₂ O ₂ production increases ( ¹⁴ ). Excessive hydrogen peroxide production has dangerous consequences for the host organisms, since hydrogen peroxide can interfere with different signaling pathways and also affects cell proliferation.

Substrates of peroxidases

Major substrates of LPO are thiocyanate (SCN ^" ) or [isothiocyanate (NCS ^" )] and iodide (F), however in the body, SCN ^" seems to be the major substrate of LPO (exocrine secretions contain much higher concentrations of SCN ^" than I ^" or NCS ^" ). Thiocyanate is oxidized into hypothiocyanate in the following lactoperoxidase catalyzed reaction:

SCN ^" (or NCS ^" or I ^" ) + H ₂ O ₂ → OSCN ^" (or ONCS ^" or OF) + H ₂ O

Even though isothiocyanate is apparently not a major substrate of LPO in the human body, the enzyme clearly capable of oxidizing also this ion similarly to oxidization of thiocyanate. This statement is e.g. supported by the fact that on 27 February 2003, FSANZ (Food Standards of Australia and New Zealand) gazetted the following amendments to the Food Standards Code: "Approval has also been given for the use of lactoperoxidase and sodium (and potassium) isothiocyanate as processing aids on the surface of meat to inhibit bacteria [approval of processing aids (A404) (A458)]" It is widely accepted that the substrate of MPO is primarily chloride, which is oxidized into hypochlorous acid in the following reaction:

Cl ^" + H ₂ O ₂ → OCl ^" + H ₂ O

It is important to mention that thiocyanate is also a substrate of MPO and SCN ^" ions seems to compete with chloride in the MPO-catalyzed reactions ( ¹⁵ ). In the plasma or interstitial fluid, where the concentration of chlo-

ride is high chloride is the dominant substrate of MPO, however in exocrine secretions (where chloride is low) thiocyanate may dominate. Hypochlorous acid and hypothiocyanate are both potent antimicrobial substances against a wide range of Gram positive and negative bacteria. Hypochlorous acid is a more effective weapon that hypothiocyanate, but it is also more toxic to the host organism ( ¹⁶ ). Beside the oxidation of halids/pseudohalids, peroxidases also effectively catalyze a different kind of biochemical reaction. They can cross-link proteins through tyrosine residues in a reaction where two tyrosine rings become covalently attached, thus forming dityrosine residues. In lower species this biochemical reaction is used for the stabilization of the extracellular matrix (such as the stabilization of cuticle in C. elegans and formation of the fertilization envelope during sea urchin fertilization) ( ¹⁷ ' ¹⁸ ). The significance of the dityrosine formation by peroxidases in the human body is unknown, however it is possible that this reaction is involved in the formation of large molecular complexes in mammalian organisms as well, which complexes may well have an adverse effect in different inflammatory disorders.

Thiocyanate (SCW) [and isothiocyanate (NCSϊ)]

Thiocyanate ions are ubiquitously present in several tissues and secretions. Thiocyanate is formed in the body during the detoxification of cyanide by rhodanase and thiocyanate is also present in food, especially in cabbage, cauliflower, etc. The concentration of SCN ^" in the human serum is 50-200 μM, and in the human saliva 500-2000 μM. The serum (and salivary) level of SCN ^" is dependent on the dietary intake of the anion and on the smoking habit of the individual. Smokers have much higher SCN ^" concentration in their blood (up to 300 μM), which is the result of increased cyanide uptake by smoking and its conversion into SCN ^" ( ¹⁹ ). Thiocyanate is en- riched in the saliva through the action of the Na-iodide symporter, which is also responsible for the accumulation of iodide into the thyroid gland ( ²⁰ ). Since SCN ^" and I ^" compete at the transporter, SCN ^" can inhibit the uptake of iodide into the thyroid gland leading to the inhibition of thyroid hormone biosynthesis. This inhibition frequently occurs in individuals who consume thiocyanate-rich food while their iodide uptake is low.

SUMMARY OF THE INVENTION

The object of this invention is, therefore, to elaborate novel uses and methods for the treatment and prevention of ulcerative colitis involving the activation of the lactoperoxidase based host defense system.

The invention is based on our original finding that LPO is expressed in the epithelium of the colon. This finding together with the previous, and so far not at all interpreted, observation present in the art that heavy smokers tend to develop ulcerative colitis shortly after abandoning smoking (whilst they are seemingly protected against the same disease while still smoking) led the present inventors to the surprising conclusion that the LPO/thiocyanate system might inhibit the development of the disease. This conclusion is further strengthened by the also known observation that serum and saliva thiocyanate levels of heavy smokers are significantly higher than normal. In accordance with the above, the present disclosure is the first to demonstrate the protecting effect of thiocyanate ions in a relevant animal model of colitis (i.e. the dextran sodium sulfate (DSS) induced colitis model in mice).

DETAILED DESCRIPTION OF THE INVENTION

The properties of thiocyanate (as well as isothiocyanate) ions outlined above, as well as our experiments being convincingly successful in the treatment of DSS induced colitis in mice, make these anions promising candidates in the treatment of ulcerative colitis. Based on our above detailed original findings and presented argu- ments, we believe that it is highly probable that higher thiocyanate levels in the plasma of smokers is responsible for the beneficial effect of smoking against colon inflammation where at least three enzymes can produce ROS (Noxl, Duox2 and Nox2) ( ²¹ ). Smokers have higher thiocyanate levels because they inhale cyanide during smoking, which is then metabolized to thiocyanate through sulfuration with thiosulfate by mitochondrial rho- danase ( ¹⁹ ). (Since no beneficial effect of smoking was described in patients with Crohn's disease, enhancing thiocyanate serum concentrations might not be beneficial in this specific disorder).

Elevating serum and/or specifically colon SCN ^" levels of patients having ulcerative colitis or individuals having a risk of developing ulcerative colitis (especially heavy smokers after abandonment of smoking) probably helps the body in fighting this specific inflammation via at least two different mechanism. On the one hand, elevated SCN ^" serum and/or colon level boosts the LPO mediated host protection system against toxic ROS com- pounds and, on the other hand, also activates the LPO mediated antimicrobial self-protection system ( ²² ). Elevated serum SCN ^" and/or colon levels can also reduce the potentially harmful tyrosine based crosslinking of proteins that may also play a role in the development of the inflammatory condition in the colon. Furthermore, elevating serum and/or colon SCN ^" levels can also reduce the production of harmful ROS by other peroxidases expressed in the colon. The early stage treatment (or prevention) of ulcerative colitis by SCN ^" therapy may be especially important, because the destruction of the epithelium in later stages of the disease might interfere with LPO expression, which seems to be essential in the effectiveness of the therapy.

Another advantage is the SCN ^" (or NCS ^" ) therapy proposed herein is the well known very low toxicity of the proposed effective agent. It is suggested that regular, daily intake of several milligrams of SCN ^" would be safe if thyroid status is monitored regularly and iodide uptake is sufficient. Addition of 40-50 mg SCN- to 1 1 of milk is a common practice in tropical areas and nobody reported about toxic effect of such milk preparations. Thiocyanate was also extensively used as a medicine earlier in the last century but, controversially, toxicity has eliminated its use. Toxicity, however, was clearly due to the fact that extremely large doses of thiocyanate were applied. Drugs containing thiocyanate included: Asthmolytan (VEB Pharmamed Naumburg), Mucidan (KaIi- Chemie, Hannover/BRD), Rhodapurin, Rhodasept C. Thiocyanate containing drugs were used to treat asthma (although in Asthmolytan ephedrine seems to be the effective component), hypertension, inflammation of the oral cavity, pharingitis and were used in antiseptic formulations.

Serum and/or colon SCN ^" (or NCS ^" ) concentrations may be advantageously elevated by the administration of SCN (or NCS) compounds (advantageously pharmaceutically acceptable salts) via the oral or parenteral route. Other sources of serum thiocyanate might include glucosinolates, which are present in many plants (especially in the family of Brassicaceae). Other compounds (e.g. nitriloside) the metabolic conversion of which provides for the formation of thiocyanate ions might also be effective in elevating serum SCN ^" concentration. Generally, a person skilled in the pertinent art can easily determine whether a candidate compound is suitable for elevating the thiocyanate or isothiocyanate ion concentrations in the serum or colon of a subject to be treated in accordance

with the present invention. Such compounds obviously include pharmaceutically acceptable thiocyanate or iso- thiocyanate salts, and compounds like different glucosinolates and nitriloside, the metabolic conversion of which is known to be resulting in the elevation of serum thiocyanate levels.

The present invention, therefore, provides for a compound the dissolution or metabolic conversion of which provides for the formation of thiocyanate or isothiocyanate ions, for use in the prevention or treatment of ulcerative colitis. The compound of the invention is advantageously selected from the group consisting of thiocyanate and isothiocyanate salts, glucosinolate compounds and nitriloside. The compound of the invention is advantageously used as an active ingredient of a pharmaceutical composition or a food additive.

The invention further provides for the use of a compound, the dissolution or metabolic conversion of which provides for the formation of thiocyanate or isothiocyanate ions, for producing a pharmaceutical composition or a food additive suitable for the treatment or prevention of ulcerative colitis. The used compound is advantageously selected from the group consisting of thiocyanate and isothiocyanate salts, glucosinolate compounds and nitriloside.

The invention also concerns pharmaceutical compositions or food additives for use in the prevention or treatment of ulcerative colitis, wherein said pharmaceutical composition or food additive comprises a compound the dissolution or metabolic conversion of which provides for the formation of thiocyanate or isothiocyanate ions.

The pharmaceutical compositions or food additives produced or applied in accordance with the invention are advantageously formulated for oral or parenteral administration and oral compositions according to the in- vention are advantageously optimized for releasing thiocyanate or isothiocyanate ions predominantly in the colon, thereby specifically providing for the elevation of the concentration of thiocyanate or isothiocyanate ions in the colon. For persons skilled in the pertinent art it is obvious how to provide oral compositions with coatings ensuring the release of the effective ingredients mostly in the colon (see e.g. the well established art pertaining to the production of the so called "enteric coated" oral compositions), specifically providing thereby for e.g. the elevation of the concentration of thiocyanate or isothiocyanate ions therein.

The invention also enables the production of pharmaceutical compositions optimized for preventive administration to subjects who abandoned smoking and, therefore, are in enhanced risk of developing ulcerative colitis.

The invention also concerns a method for producing a pharmaceutical composition or a food additive suitable for the prevention or treatment of ulcerative colitis, said method comprising the mixing of a compound the dissolution or metabolic conversion of which provides for the formation of thiocyanate or isothiocyanate ions with pharmaceutically acceptable carriers and/or other additives.

The invention further provides for a method of treating or preventing ulcerative colitis comprising administering an effective amount of a composition comprising, as an active ingredient, a compound the dissolution or metabolic conversion of which provides for the formation of thiocyanate or isothiocyanate ions, to a patient suf- fering from or having a risk of developing ulcerative colitis. In the method of the invention, the administration of compositions comprising compounds the dissolution or metabolic conversion of which provides for the formation of thiocyanate or isothiocyanate ions is advantageously done via the oral or parenteral route.

The invention also concerns a method for preventing the onset of ulcerative colitis in individuals who abandoned smoking.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the results of a Northern blot experiment performed on colon total RNA isolates, demonstrating the expression of LPO mRNA in both mouse and rat colon (the 957 bp Dra III fragment of the mouse LPO cDNA was used as an LPO specific probe).

Fig. 2 is a graph showing the effect of SCN treatment on the disease activity index (DAI) of mice having DSS-induced colitis.

Fig. 3 is a graph showing the effect of SCN treatment on DSS-induced mortality in mice.

Fig. 4 is a graph showing the effect of SCN treatment on colon length reduction induced by DSS in mice.

EXAMPLES

Example 1

The expression of LPO mRNA in the mouse and rat colon

The expression of LPO mRNA in the mouse and rat colon was studied by Northern blot analysis. A 957 bp fragment of the mouse LPO cDNA was generated by Dra III cut. Radioactive labeling was performed by Prime- It® Random Primer Labeling Kit (Stratagene) using [α- ³² P]dCTP. For analysis of tissues, total RNA was prepared from mouse and rat colon, electrophoretically separated on a 1% agarose formaldehyde gel and transferred to nylon membrane. Membranes were probed at 60 ⁰ C by standard hybridization protocols. As can be seen on Fig. 2, both rat and mouse colon RNA isolates proved to be positive for LPO mRNA expression. This so far unprece- dented finding, together with the known observations concerning the relative protection of heavy smokers against ulcerative colitis and the high serum thiocyanate levels of heavy smokers, led the present inventor to the conclusion that the LPO/thiocyanate system is associated with the disease ulcerative colitis and, therefore, thiocyanate and iso thiocyanate ions should be useful in the treatment and prevention of the said disease.

Example 2

Protective effect of thiocyanate (SCN) in the DSS-induced model of ulcerative colitis in mice

Background

Dextran sodium sulfate (DSS) polymers in drinking water induce colitis in mouse. The mechanism of DSS action is still not fully understood, but it is likely that DSS damages the colon epithelium and this primary damage is followed by an acute inflammatory response. Studies on knock-out animals (including experiments with T- and B-cell-deficient animals) showed that the adaptive immune system does not play a role in the development of the disease. Therefore the DSS colitis model is well-suited for studying the role of innate immunity in the development of colitis. Although no perfect animal model of ulcerative colitis currently exists the DSS-induced model seems to be ideally suited for studying the possible therapeutic effect of thiocyanate since peroxidases are components of the innate immune defense mechanisms. Experimental Protocol

We used 8-10 weeks old C57/BL6 mice in our experiments. We applied 5 % DSS in the drinking water of the animals for 7 days and fresh DSS containing water was added at day 4. During the experiment we measured the weight of the animals every day and we also analyzed the stool consistency and the presence of blood. These

data along with score of the animals' behavior were merged into a clinical score according to Van der Sluis et α/.( ²³ ) (see Table 1). At the end of the experiments the animals were sacrificed and the entire colon was removed. We measured the length of the colon, which is another sensitive indicator of the severity of the disease. Pieces of colon tissue were removed and processed for immunohisto chemistry.

Table 1 Disease Activity Index (DAI) Score

Administration of Thiocyanate

In the beginning of our experiments we added thiocyanate in the drinking water, however later, in order to achieve a better-controlled dosing we started to administer thiocyanate through orogastric tube. The dosing was as follows: 5 mg/kg/day. We also tried higher (5X) and lower (1/5 X) doses of thiocyanate. In the control experiments we applied the same dose of NaCl in drinking water.

Measurement of Serum Thiocyanate

The efficacy of thiocyanate administration was controlled by serum thiocyanate measurements. We pre- pared a stock sodium thiocyanate standard of 1000 μmol/L and working standards of 50, 100, 150, 200 and 250 μmol/L respectively. In a small Eppendorf tube we mix the followings: 50 μl standard or sample, 25 μl 0.1 M sodium perchlorate solution, 25 μl 20 % TCA solution.

After mixing we incubated the sample at room temperature for 20 minutes, then we centrifuged down the precipitated protein. We took the supernatant and mixed with 50 μl ferric nitrate 20 g/1 solution. We measured the absorbance in a microplate reader at 455 nm.

After measuring the standards we made a calibration curve and thiocyanate concentrations of the serum sample were determined.

Results

As shown in Figure 2, administration of thiocyanate resulted a clear reduction in DAI when compared to control, NaCl-treated animals.

Furthermore thiocyanate treatment reduced the mortality induced by DSS treatment (see Figure 3).

We also measured the colon length at the end of the 7 days period of DSS treatment. As shown in Figure 4, colons from SCN-treated animals were significantly longer when compared to control (NaCl-treated animals).

Conclusion Our experiments clearly demonstrate that SCN treatment effectively reduces the symptoms of DSS-induced colitis. These results strongly suggest that compounds providing for the elevation of serum or colon SCN ^" (or NCS ^" ) levels should also be effective in the treatment of ulcerative colitis.

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